Inverted Fluorescence Microscope

What is the Inverted Fluorescence Microscope?

An inverted fluorescence microscope illuminates and observes live cells from below through the base of the culture vessel. It is the standard configuration for fluorescence imaging of living cells in dishes, flasks and multi-well plates.

Overview

An inverted fluorescence microscope places the objective below the stage and the illumination above, so the specimen remains in its culture vessel during observation. Long working distance objectives accommodate the thickness of the plastic base and allow access for micromanipulation, perfusion and temperature control. Episcopic fluorescence illumination passes through the objective as in the upright design, and filter cubes provide channel selection. Environmental chambers maintain temperature and carbon dioxide for live-cell work, and motorised stages support time-lapse imaging. The configuration is essential for cell biology, where cells must remain viable and in medium throughout the experiment.

How does it work?

A fluorescence microscope illuminates the specimen with a selected excitation wavelength using a filter cube containing an excitation filter, a dichroic mirror and an emission filter. Fluorophores in the specimen absorb the excitation light and emit at longer wavelengths. The dichroic mirror reflects the excitation light onto the specimen and transmits the emitted light to the objective and detector, so background illumination is largely excluded. The specimen therefore appears bright against a dark background, giving high contrast and specificity. Multiple filter sets allow different fluorophores to be imaged in turn, and excitation and emission bands must be chosen to avoid overlap between channels. Bright sources, efficient filters and high-numerical-aperture objectives determine sensitivity.

What are the key features?

• Objective below the stage for vessel-based imaging
• Long working distance objectives
• Episcopic fluorescence with filter cubes
• Environmental chamber for live-cell conditions
• Motorised stage option for time-lapse imaging

What are the technical specifications?

A typical inverted fluorescence microscope configuration offers optical system of Infinite corrected, fluorescence, filter cubes of DAPI, FITC, TRITC, 4-position turret, light source of LED or 100 W mercury lamp, objectives of 4×, 10×, 20×, 40× long working distance, working distance of Up to 8 mm and stage of Fixed or motorised, for dishes and plates. Values are indicative of the product class; the exact figures for each configuration are confirmed on the quotation and the calibration documents supplied with the instrument.

Which applications is it used for?

The Inverted Fluorescence Microscope is used in live-cell fluorescence imaging, time-lapse microscopy of cultured cells, multi-well plate screening and micromanipulation and microinjection. Across the wider Fluorescence Microscopes class, laboratories also deploy it for immunofluorescence and cell biology, fluorescence in-situ hybridisation and microbiology and viability staining.

Which standards and certifications apply?

Relevant standards and methods include ISO 8036, ISO 19012-1, ISO 9345-1, ISO 8255-1 and IEC 61010-1. Certification candidates for this model are CE and ISO 9001 manufacturing; each is to be confirmed for the specific configuration before shipment, and certificates are supplied only where they have been issued.

Protect the specimen from unnecessary light to reduce photobleaching, and use the lowest excitation intensity that gives adequate signal. Select filter sets that match the fluorophores and avoid spectral overlap between channels. Keep filters, dichroic mirrors and objectives clean, since contamination causes background fluorescence and scattering. Mercury and xenon lamps need warm-up, consistent alignment and careful disposal, while light-emitting diodes are stable and long-lived. Verify background and uniformity with a blank slide before imaging. Record excitation and emission settings with the images for reproducibility, and maintain the camera and software for quantitative work.

How do I select the right Inverted Fluorescence Microscope?

Choose an inverted fluorescence microscope for live cells in vessels or for time-lapse work. Confirm the working distance and objective correction suit the vessel base thickness, and that environmental control maintains viability. For multi-well or multi-position imaging, specify a motorised stage and software for automated acquisition. Match filter cubes to the fluorophores used.

How does it compare with other Fluorescence Microscopes variants?

Choose a fluorescence microscope when specific molecules must be detected through fluorescent labels or natural autofluorescence. Match the filter sets to the fluorophores in use, such as DAPI for nuclei or FITC for green emission, and confirm the excitation source covers all required wavelengths. Decide between an upright configuration for slides and sections and an inverted one for live cells in dishes. For optical sectioning and three-dimensional imaging, a confocal system is required. Consider LED sources for stability and low running cost versus arc lamps for very high intensity. Check objective numerical aperture, transmission and correction, and the camera sensitivity for the emission wavelengths used.